Antigen polypeptide for the diagnosis and/or treatment of ovarian cancer

ABSTRACT

The invention provides an isolated antigen polypeptide that can be expressed in a subject with ovarian cancer. Also provided is the diagnosis of ovarian cancer by using the antigen polypeptide of the invention and the prevention and/or treatment of ovarian cancer by suppressing the gene of the antigen polypeptide of the invention.

FIELD OF THE INVENTION

The invention provides an isolated antigen polypeptide that can beexpressed in a subject with ovarian cancer. Also provided is thediagnosis of ovarian cancer by using the antigen polypeptide of theinvention and the prevention and/or treatment of ovarian cancer bysuppressing the gene of the antigen polypeptide of the invention.

BACKGROUND OF THE INVENTION

Ovarian cancer is the leading cause of cancer deaths in women worldwideand causes more deaths than all other gynecologic malignancies combined.All women are at risk for ovarian cancer, but older women are morelikely to get the disease than younger women. About 90 percent of womenwho get ovarian cancer are older than 40 years of age, with the greatestnumber being 55 years or older. Although ovarian cancer remains thenumber one killer of women with gynecologic malignant hyperplasia, whenovarian cancer is found in its early stages, treatment is mosteffective. However, ovarian cancer does not cause many symptoms in itsearly stages. This is why most cases are not found until the cancer hasspread. Ovarian cancer at its early stages (I/II) is difficult todiagnose; when it spreads and advances to later stages (III/IV),diagnosis is easier. This is due to the fact that most of the commonsymptoms are non-specific. Approximately 75% of women diagnosed withsuch cancer are already at the advanced stages (III and IV) of thedisease at their initial diagnosis.

The serum BHCG level should be measured in any female in whom pregnancyis a possibility (Christoph Steinmeyer, Tumor Biol 2003; 24:13-22).Moreover, serum alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH)should be measured in young girls and adolescents with suspected ovariantumors because the younger the patient, the greater the likelihood of amalignant germ cell tumor. However, outside the aforementionedcollection of substances, there has been a relative dearth of antigenswhich are useful in diagnosis and monitoring; this has been provenparticularly true with respect to gynecologic malignancies, especiallyovarian carcinomas, which frequently spread throughout the pelvic cavitybefore diagnosis of the condition. Many of these carcinomas typicallyexhibit a very aggressive growth pattern and generally respond well tochemotherapy. Thus, an accurate method by which early diagnosis of thesediseases could be obtained is highly desirable.

A discovery (Bast, et al., N. Engl. J. Med. 309: 883 [1983]) of a serouscystadinocarcinoma ovarian antigen, known as CA125, has been found to beof significant value in monitoring patients with ovarian cancer. Thisantigen was isolated by using a monoclonal antibody, OC125, made bystimulation of mice with ovarian cancer cell line OVCA 433. It has beenshown to recognize cell surface antigens of the OVCA 433 cell as well as13 of 14 other ovarian cancer cell lines and a melanoma cell line. Theantigen is a high molecular weight (>200,000 daltons) glycoprotein whichhas been partially purified from tissue culture medium (Masuko, et al.,Cancer Res. 44: 2813, 1984). Furthermore, U.S. Pat. No. 4,921,790relates to a 40 kilodalton subunit of serous cystadinocarcinoma ovariantumor associated antigen CA125, useful in the diagnosis and monitoringof ovarian cancer. Although a blood test called CA-125 is useful indifferential diagnosis and in follow-up on the disease, it has not beenshown to be an effective method of screening for early-stage ovariancancer due to its unacceptably low sensitivity and specificity. Elevatedlevels of serum CA125 alone or in combination with other knownindicators, however, do not provide a definitive diagnosis ofmalignancy, or of a particular malignancy such as ovarian carcinoma.

Current research is looking at ways to combine tumor marker proteomicswith other indicators of disease (i.e., radiology and/or symptoms) inorder to improve accuracy. The challenge in such an approach is that thevery low population prevalence of ovarian cancer means that even testingwith very high sensitivity and specificity will still lead to a numberof false positive results (i.e., performing surgical procedures in whichcancer is not found). However, the contributions of proteomics are stillin the early stages and require further refining. Current studies onproteomics mark the beginning of a paradigm shift towards individuallytailored therapy. Currently, neither diagnosis nor five-year survivalhas greatly improved for these patients. This is substantially due tothe high percentage of advanced-stage initial detection of the disease.Therefore, the challenge of developing new detection technology toimprove early diagnosis and reduce the percentage of advanced-stageinitial diagnoses still exists.

SUMMARY OF THE INVENTION

The invention provides an isolated antigen polypeptide expressed in asubject with ovarian cancer, selected from the group consisting of: (a)a polypeptide comprising an amino acid sequence having at least 85%sequence identity with SEQ ID NO:2; (b) a polypeptide that is encoded bya polynucleotide that hybridizes under stringency conditions with SEQ IDNO: 1 or a full-length complementary strand of SEQ ID NO: 1; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 85% sequence identity with SEQ ID NO: 1; and (d) apolypeptide comprising an amino acid fragment encoded by apolynucleotide as shown in SEQ ID NO: 4 or comprising an amino acidfragment as shown in SEQ ID NO: 3, provided that the sequence of thepolypeptide is included within (a), (b) or (c).

The invention also provides a polynucleotide encoding the isolatedantigen polypeptide of the invention.

The invention further provides an antibody specifically binding to asequence comprising at least the consensus sequence X₁-P-H-X₂-Y-X₃-X₄contained in the C-terminal of the antigen polypeptide of the invention.

The invention further provides a kit for the detection of ovarian cancerin a subject comprising the antibody of the invention.

The invention further provides a method for the diagnosis of thepresence of an ovarian cancer in a subject comprising: detecting theexpression of the antigen polypeptide of the invention in a biologicalsample from a subject, under conditions and for a time sufficient todetect the said expression, wherein the expression of the said antigenpolypeptide represents the presence of an ovarian carcinoma and theover-expression of the said antigen polypeptide represents the presenceof not only an ovarian cancer but also metastasis of the ovarian cancer.

The invention also further provides a method for the diagnosis ofovarian cancer in a subject comprising contacting a biological samplefrom a subject with the antibody of the invention, to determine thepresence in the biological sample of the antigen polypeptide of theinvention, under conditions and for a time sufficient to detect bindingof the antibody to the consensus sequence, wherein the bindingrepresents the presence of an ovarian cancer. The invention also furtherprovides a method for the prevention and/or treatment of ovarian cancer,comprising the step of suppressing the expression of the antigenpolypeptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows serological identification of ovarian cancertumor-associated peptides. Serological identification of ovariantumor-associated antigen genes was carried out using a modified phagedisplay technology. An M13 phage library was applied in which each phagedisplayed various 7-mer random peptides. The library was pre-absorbedwith immunoglobulin IgGs purified from the sera of 30 healthy donors,including 15 males and 15 females, to mask and remove immunogenicepitopes recognized by the normal antibodies. Thereafter, the unboundphages were panned against antibodies purified from 32 patients withovarian cancer. Bio-panning screening to enrich phages displaying thetumor-associated peptides is schematically shown in FIG. 1. Selection ofpotential tumor-associated peptides from the resulting library wasperformed by coating with individual purified ascetic or serum antibodyin each well of ELISA plates.

FIG. 2 shows the binding of the phage containing Peptide #10 toantibodies purified from 11 patients with ovarian cancer in ELISA assay(1-12: Ig purified from 12 patients as coating antigens for detectingphage #10; 11c: Ig purified from patient 11 as coating antigen fordetecting M13 phage as a control experiment; nIg: Ig purified from 30normal subjects as coating antigen for detecting phage #10; and nS:normal sera as coating antigen for detecting phage #10). ELISA assay wasperformed by coating the purified antibody in each well. The boundphages were detected with an antibody specific to M13 phages andvisualized by incubating with ABTS, an HRP substrate in the assay. Forbackground control, purified or unpurified serum antibodies or bovineserum albumin were coated onto separated wells. In addition, M13 phagewithout displaying any peptide was included as a negative control. Theresults show that the phage with Peptide #10 specifically binds to theantibody purified from patient 11 (CA502).

FIG. 3 shows the confirmation of OVTA1 as an antigenic target of CA502antibodies. To confirm that OVTA1 was an antigenic target of CA502antibody, C-terminus (CT) of the putative gene was cloned out from ahuman fetal brain cDNA library, expressed as an myc-tagged protein inHeLa cells, and probed with CA502 antibody. Western blot analysis shownin FIG. 3A clearly demonstrated positive immunoreactivity of CA502antibody to the C-terminus that contains Peptide #10 sequence, TPHGYAH.To further examine whether this peptide is an immunogenic epitope ofCA502 antibody, site-directed deletion mutagenesis was performed.Deletion of the peptide from the C-terminus resulted in loss ofimmunoreactivity of CA502 antibody (FIG. 3B), indicating that therandom-peptide phage displaying system is a powerful tool for screeningimmunogenic epitope(s) of an antigen.

FIG. 4 shows the determination of sizes of OVTA1 gene transcript inovarian cancer cells. Determination of sizes of OVTA1 gene transcript(s)was carried out by Northern analyses of ovarian cancer cells. The totalRNAs extracted from SK-OV-3, OVCAR3, and TOV-112D cells were resolved ona denaturing formaldehyde-containing gel. An antisense sequencespecifically primed on OVTA1 gene transcript was radiolabelled with[α-32 P]dATP and hybridized with the extracted RNA of those cells. InFIG. 4, four radiolabelled bands in total were detected, and it wasfound that 4.5-kb transcript was the major one in all the ovarian cancercell lines tested.

FIG. 5 shows that the putative KIAA0999 gene published in NCBI databasecould be translated into a 150 KDa-polypeptide chain. In Westernblotting analyses using a rabbit antiserum made from the C-terminus ofOVTA1, ovarian cancer cells consistently showed four protein bands (FIG.5). A major protein around 150 KDa is found in all the cells, suggestingthat the coding sequence of OVTA1 gene is approximately 4.0 Kb.

FIG. 6 shows that OVTA 1 universally existed in multiple tissues.

FIG. 7 shows the over-expression of OVTA1 in OVCAR-3 cells.

FIG. 8 shows that the over-expression of OVTA1 significantly increasescell proliferation and cell migration in OVCAR3 cells. FIGS. 8A and 8Bshow that the over-expression of OVTA1 in OVCAR3 cells significantlyincreased cell proliferation rate and cell migration (metastasis) byaround 2.75 to 3.1 times, respectively, compared to control (FIG. 8B).

FIG. 9 shows the knockdown of OVTA1 in SKOV-3 cells.

FIG. 10 shows that the knockdown of OVTA1 significantly reduced cellgrowth and cell migration in SKOV-3 cells. FIGS. 10A and 10B show thatthe suppression of OVTA1 expression in SK-OV3 cells significantlyinhibited cell proliferation rate and cell migration (metastasis),respectively.

FIG. 11 shows that OVTA1 expression is correlated with tumorprogression. FIG. 11A shows that tumors developed in all the micesubcutaneously inoculated with SK-OV3 cells but not in the miceinoculated with SK-OV3/shOVTA1 cells. FIG. 11B shows that the knockdownof OVTA1 decreases tumor growth rate in NOD-SCID mice.

FIG. 12 shows the immunohistochemic results that clearly demonstratethat OVTA1 is over-expressed in ovarian cancer tissues (e) compared tonormal ovarian tissues (a) or tissues from adenomyosis (c). In contrast,CA-125 antigen can be detected in both ovarian cancer tissues (f) andtissues from adenomyosis (d). In (b), the normal ovarian tissue as thecontrol for CA-125 antigen detection is shown.

DETAILED DESCRIPTION OF THE INVENTION

The lack of good diagnostic markers allowing early detection of thedisease is one of the causes of the low survival rate of ovarian cancerpatients. Further compounding this difficulty in early diagnosis is thelack of effective treatments for ovarian cancer, development of whichhas been impeded by a deficit in the general understanding of ovariancancer biology. The invention overcomes these deficits in the art byproviding an ovarian tumor marker (OVTA1) that is expressed by OVTA1gene. The diagnosis of ovarian cancer using OVTA1 of the inventionpossesses high specificity and sensitivity. Furthermore, the knockdownof OVTA1 can inhibit ovarian tumor cell growth and migration, suggestingthat it can be a target for treating ovarian cancer.

Definitions

The term “variant” as used herein means an isoform, an allelic variantof a gene or a region thereof, a naturally occurring mutant form of agene or a region thereof, or a polypeptide or fragment having an aminoacid sequence that differs in one or more amino acids but which retainsone or more aspects of the desired characteristic biological function.This may be achieved by the addition of one or more amino acids to anamino acid sequence, the deletion of one or more amino acids from anamino acid sequence and/or the substitution of one or more amino acidsfor another amino acid or amino acids. Inversion of amino acids and anyother mutational change that results in alteration of an amino acidsequence are also encompassed. A variant may be prepared by introducingnucleotide changes in a nucleic acid sequence so that the desired aminoacid changes are achieved upon expression of the mutated nucleic acidsequence, or, for instance, by synthesizing an amino acid sequenceincorporating the desired amino acid changes, which is well within thecapability of the skilled persons.

The term “OVTA1 gene” as used herein means the full-length gene withnucleotides 438 to 4229 (SEQ ID NO:1) of the sequence that is locatedunder Genbank accession number: AB023216. This sequence “AB023216” andits protein product “KIAA0999” (Product ID: 06551) were disclosed in DNARes. 1999 Feb. 26; 6(1):63-70, whereas its coding gene sequence, thefunctional protein encoded therefrom and their functions have not beenidentified yet.

The term “OVTA1 protein” as used herein means the whole sequence of theprotein encoded by the OVTA1 gene and its amino acid sequence is shownin SEQ ID NO:2.

The term “isolated” as used herein means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring polypeptide orpolynucleotide present in a living animal is not isolated, but the samepolypeptide or polynucleotide, separated from some or all of theco-existing materials in the natural system, is isolated. Suchpolypeptides or polynucleotides can be part of a composition and stillbe isolated if such composition is not part of its natural environment.

The term “polypeptide” as used herein means a linear series of aminoacids connected to one another by peptide bonds between the alpha-aminoand carboxy groups of adjacent amino acids.

The term “polynucleotide” or “nucleotide” means polynucleotidescomprising DNA. The polynucleotides of the embodiments also encompassall forms of sequences including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

The terms “encoding” and “encoded” mean that the polynucleotide ornucleic acid comprises the requisite information for direct translationof the nucleotide sequence into a specified protein. The information bywhich a protein is encoded is specified by the use of codons. A nucleicacid or polynucleotide encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “ovarian cancer” as used herein includes primary and metastaticovarian carcinoma. Criteria for classification of a malignancy asovarian carcinoma are well known in the art (see Bell et al., 1998 Br.J. Obstet. Gynaecol. 105:1136; Meier et al., 1997 Anticancer Res.17(4B):3019; Cioffi et al., 1997 Tumori 83:594) as are the establishmentand characterization of human ovarian carcinoma cell lines from primaryand metastatic tumors (e.g., OVCAR-3, Amer. Type Culture Collection,Manassas, Va.; Yuan et al., 1997 Gynecol. Oncol. 66:378).

The term “antibodies” as used herein includes polyclonal antibodies,monoclonal antibodies, and fragments thereof as well as any naturallyoccurring or recombinantly produced binding partners, which aremolecules that specifically bind the antigen polypeptide of theinvention. Antibodies are defined as “immunospecific” or specificallybinding if they bind the antigen polypeptide of the invention with ahigh affinity. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al., Ann. N.Y. Acad. Sci. 51:660 (1949).

The term “expression” as used herein includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The term “expression vector” as used herein refers to a linear orcircular DNA molecule which comprises a polynucleotide encoding apolypeptide of the invention, and which is operably linked to additionalnucleotides that provide for its expression. The term “operably linked”denotes a configuration in which a control sequence is placed at anappropriate position relative to the coding sequence of thepolynucleotide sequence so that the control sequence directs theexpression of the coding sequence of a polypeptide.

The term “host cell” as used herein includes any cell type which issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct comprising a polynucleotide of the presentinvention.

The term “identity” as used herein refers to the relatedness between twoamino acid sequences or between two nucleotide sequences. For thepurposes of the present invention, the degree of identity between twoamino acid sequences or two nucleotide sequences is determined by theknown methods and software. For example, Clustal method (Higgins, 1989,CABIOS 5: 151-153) or the Wilbur-Lipman method (Wilbur and Lipman, 1983,Proceedings of the National Academy of Science USA 80: 726-730).

Antigen Polypeptide of the Invention and Polynucleotides Encoding theSame

The invention is the first to unexpectedly discover the antigenpolypeptide (OVTA1 protein) encoded by OVTA1 gene that can be expressedin a subject with ovarian cancer. The above-mentioned antigenpolypeptide is named an ovarian tumor-associated antigen. By detectingthe existence of the antigen polypeptide (OVTA1 protein) in a subject,the ovarian cancer can be diagnosed.

The invention provides an isolated antigen polypeptide expressed in asubject with ovarian cancer, selected from the group consisting of: (a)a polypeptide comprising an amino acid sequence having at least 85%sequence identity with SEQ ID NO:2; (b) a polypeptide that is encoded bya polynucleotide that hybridizes under stringency conditions with SEQ IDNO: 1 or a full-length complementary strand of SEQ ID NO: 1; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 85% sequence identity with SEQ ID NO: 1; and (d) apolypeptide comprising an amino acid fragment encoded by apolynucleotide as shown in SEQ ID NO: 4 or comprising an amino acidfragment as shown in SEQ ID NO: 3, provided that the sequence of thepolypeptide is included within (a), (b) or (c).

In a first aspect, the invention relates to isolated polypeptides havingan amino acid sequence which has a degree of identity to SEQ ID NO: 2 ofat least 85%, preferably at least 90%, more preferably at least 95%, andmost preferably at least 98%, and which can be expressed in a subjectwith ovarian cancer.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2, a variant thereof, or a fragment thereofthat can be expressed in a subject with ovarian cancer. According to theinvention, the variant of the invention may be artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO: 2 or the mature polypeptidethereof. Preferably, amino acid changes are of a minor nature, that is,they are conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein.Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, The Proteins, Academic Press, New York. The most commonlyoccurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

According to a further embodiment of the invention, the antigenpolypeptide of the invention comprises the amino acids shown in SEQ IDNO:2.

In a second aspect, the present invention relates to isolatedpolypeptides expressed in a subject with ovarian cancer which areencoded by polynucleotides which hybridize under stringency conditions,preferably moderate stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with nucleotides shown in SEQ ID NO: 1 or a full-lengthcomplementary strand of SEQ ID NO: 1 (J. Sambrook, E. F. Fritsch, and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition,Cold Spring Harbor, N.Y.). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Forpreliminary screening for homologous nucleic acids, low stringencyhybridization conditions, corresponding to a T^(m) (melting temperature)of 55° C., can be used, e.g., 5 times SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5 times SSC, 0.5% SDS. Moderate stringencyhybridization conditions correspond to a higher T^(m), e.g., 40%formamide, with 5 times or 6 times SCC. High stringency hybridizationconditions correspond to the highest T^(m), e.g., 50% formamide, 5 timesor 6 times SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate.

In a third aspect, the present invention relates to the isolatedpolypeptide expressed in a subject with ovarian cancer which is apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 85% sequence identity, preferably at least 90%, morepreferably at least 95%, and most preferably at least 98%, which can beexpressed in a subject with ovarian cancer, with the nucleotides shownin SEQ ID NO: 1.

According to one embodiment of the invention, the antigen polypeptide ofthe invention can be encoded by the nucleotide sequence shown in SEQ IDNO:1 or its degenerate sequence. According to a further embodiment ofthe invention, the antigen polypeptide of the invention can be encodedby the nucleotide sequence shown in SEQ ID NO:1.

In a fourth aspect, the invention relates to the isolated polypeptidesexpressed in a subject with ovarian cancer, which comprise an amino acidfragment encoded by a polynucleotide as shown in SEQ ID NO: 4 or anamino acid fragment as shown in SEQ ID NO: 3, provided that the sequenceof the polypeptide is included within (a), (b) or (c). The inventionalso unexpectedly finds that the polypeptides having an amino acidfragment 955 to 1112 (SEQ ID NO:3) of OVTA1 protein possess betterimmunogenic activity and can also be used as an ovarian tumor-associatedantigen. The above-mentioned polypeptides are encoded by the nucleotides2863 to 3336 (SEQ ID NO:4) of OVTA1 gene.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide of the invention are known in the art and include isolationfrom genomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the polynucleotides of the present invention from suchgenomic DNA can be effected by, for example, using the well-knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. Other nucleic acid amplification procedures such as ligasechain reaction (LCR), ligated activated transcription (LAT) andnucleotide sequence-based amplification (NASBA) can be used.

The antigen polypeptide of the invention may be prepared by processeswell known to those skilled in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems that comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems, andto the production of polypeptides of the invention by recombinanttechniques. Therefore, the invention also relates to nucleic acidconstructs comprising an isolated polynucleotide of the presentinvention operably linked to one or more control sequences which directthe expression of the coding sequence in a suitable host cell underconditions compatible with the control sequences. An isolatedpolynucleotide encoding a polypeptide of the present invention may bemanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the polynucleotide's sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotide sequencesutilizing recombinant DNA methods are well known in the art.

For production of the antigen polypeptides of the invention, host cellscan be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). Representative examples ofappropriate hosts include bacterial cells, such as cells of E. coli(JM109 strain).

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors. The expression systemconstructs may contain control regions that regulate as well as engenderexpression. Generally, any system or vector suitable to maintain,propagate or express polynucleotides and/or to express a polypeptide ina host may be used for expression in this regard. The appropriate DNAsequence may be inserted into the expression system by any of a varietyof well-known and routine techniques, such as, for example, those setforth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL(suppl).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

The expressed antigen polypeptide of the invention may also be useful astarget antigens in any of a number of assay configurations for routineantibody screening, which can be readily performed by those havingordinary skill in the art.

Antibodies Specific to the Antigen Polypeptide of the Invention

The invention provides an antibody specifically binding to a sequencecomprising at least the consensus sequence X₁-P-H-X₂-Y-X₃-X₄ containedin the C-terminal of the antigen polypeptide of the invention, whereinX₁, X₂, X₃ and X₄ can be any amino acid. Preferably, X₁ can be Q, N, S,T, V, W, A or L; X₂ can be H, S, G or N; X₃ can be S, P, M, F, A or Kand X₄ can be L, H, K, F, M, S or R. More preferably, the consensusmotif has the amino acid sequence Thr-Pro-His-Gly-Tyr-Ala-His (SEQ IDNO:5). The invention finds that the antigen polypeptide contains aconsensus motif, X₁-P-H-X₂-Y-X₃-X₄, which is a highly immunogenicepitope for an antibody. According to one preferred embodiment of theinvention, the antibody of the invention specifically binds to theantigen polypeptide of the invention. More preferably, the antibody ofthe invention specifically binds to SEQ ID NO:2 or SEQ ID NO: 3 of theinvention.

Antibodies that are specific for the consensus sequence of the antigenpolypeptide of the invention are readily generated as monoclonalantibodies or as polyclonal antisera, or may be produced as geneticallyengineered immunoglobulins (Ig) that are designed to have desirableproperties using methods well known in the art. The polyclonal antibodycan be obtained by purifying the obtained antiserum through an optionalcombination of known purification methods such as salting out, ionexchange chromatography, affinity chromatography and the like. Themonoclonal antibody can be obtained in the following manner. Antibodyproducing cells such as splenocytes, lymphocytes or the like arecollected from the immunized animal and fused with myeloma cells or thelike to make them into hybridoma cells by a known method in whichpolyethylene glycol, Sendai virus, an electric pulse or the like isused. Thereafter, a clone capable of producing an antibody which bindsto the consensus sequence of the antigen polypeptide of the invention isselected and cultured, and the monoclonal antibody of interest ispurified from the resulting culture supernatant. The purification may beeffected through an optional combination of known purification methodssuch as salting out, ion exchange chromatography, affinitychromatography and the like. The novel antibody can also be obtained bymeans of genetic engineering techniques. That is, mRNA is isolated fromsplenocytes or lymphocytes of an animal which is immunized with theconsensus sequence of the antigen polypeptide of the invention or from ahybridoma capable of producing a monoclonal antibody which is specificfor the consensus sequence of the antigen polypeptide of the invention,and a cDNA library is prepared using the isolated mRNA. Thereafter, aclone capable of producing an antibody which reacts with the consensussequence of the antigen polypeptide of the invention is screened fromthe cDNA library and cultured to obtain a culture supernatant from whichthe antibody of interest is purified through a combination of knownpurification methods. For example, by way of illustration and notlimitation, antibodies may include recombinant IgGs, chimeric fusionproteins having immunoglobulin derived sequences or “humanized”antibodies that may all be used for detection of the OVTA1 according tothe invention.

Kits Comprising Antibodies that Bind to a Sequence Comprising at leastthe Consensus Sequence of the Antigen Polypeptide of the Invention

The invention also provides kits comprising the isolated antigenpolypeptide of the invention or antibodies that bind to a sequencecomprising at least the consensus sequence of the antigen polypeptide ofthe invention, for use in diagnosis of ovarian cancer. Accordingly, thekits comprise an isolated antigen polypeptide of the invention or anantibody that can bind to a sequence comprising at least the consensussequence of the antigen polypeptide of the invention specifically and/orform a complex with the sequence. More preferably, the antigenpolypeptide of the invention has the amino acid sequence as shown in SEQID NO: 2 or SEQ ID NO:3 and the antibody of the invention specificallybinds to SEQ ID NO:2 or SEQ ID NO: 3 of the invention. The binding of anantibody (e.g., monoclonal, polyclonal, human, humanized, etc.) is usedfor diagnosing ovarian cancer in an individual. The kits of thisinvention are in suitable packaging, and optionally provide additionalcomponents such as buffers and instructions for determining binding to asequence comprising at least the consensus sequence of the antigenpolypeptide of the invention, such as capture reagents, developingreagents, labels, reacting surfaces, means for detection, controlsamples, and interpretive information. The instructions may be for anymeasurement of antigen binding, including, but not limited to, thoseassays described herein. In some embodiments, reagents described aboveare supplied so that multiple measurements may be made, for example,allowing for measurements in the same individual over time or multipleindividuals. Any appropriate means for detecting binding of theantibodies may be employed (and provided in the kits), such as a labeledanti-human antibody, wherein the label may be an enzyme, fluorophore,chemiluminescent material radioisotope or coenzyme. Generally, the labelused will be an enzyme.

Methods for Detecting or Diagnosing Ovarian Cancer in a BiologicalSample

The invention provides a method for the diagnosis of the presence of anovarian cancer in a subject comprising: detecting the expression of theantigen polypeptide of the invention in a biological sample from asubject, under conditions and for a time sufficient to detect the saidexpression.

The invention also provides a method for the diagnosis of ovarian cancerin a subject comprising contacting a biological sample from a subjectwith at least one antibody specific for a sequence comprising at leastthe consensus sequence X₁-P-H-X₂-Y-X₃-X₄ (preferablyThr-Pro-His-Gly-Tyr-Ala-His) contained in the C-terminal of the antigenpolypeptide of the invention, to determine the presence in thebiological sample of the antigen polypeptide of the invention, underconditions and for a time sufficient to detect binding of the antibodyto the consensus sequence, and therefrom detecting the presence of anovarian cancer or metastasis of the ovarian cancer. The expression ofthe said antigen polypeptide represents the presence of an ovariancarcinoma and the over-expression of the said antigen polypeptiderepresents the presence of not only an ovarian cancer but alsometastasis of the ovarian cancer.

According to the invention, the antigen polypeptide of the invention maybe detected in a biological sample from a subject or biological source.Biological samples may be provided by obtaining a blood sample, biopsyspecimen, tissue explant, organ culture, biological fluid or any othertissue or cell preparation from a subject or a biological source. Thesubject or biological source may be a human or non-human animal, aprimary cell culture or culture adapted cell line including but notlimited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid celllines, differentiated or differentiatable cell lines, transformed celllines and the like. In certain preferred embodiments of the invention,the subject or biological source may be suspected of having or being atrisk for ovarian cancer, and in certain other preferred embodiments ofthe invention the subject or biological source may be known to be freeof a risk or presence of such disease. According to the invention, thebiological sample is selected from the group consisting of ovariantissue, ovarian cell, blood, serum, plasma, ascites fluid, andperitoneal fluid.

According to the invention, the over-expression of the antigenpolypeptide of the invention or OVTA1 protein can significantly increaseproliferation and migration of ovarian cancer cells. Therefore, thedetection of the expression level of the antigen polypeptide of theinvention can be used to diagnose ovarian cancer and its metastasis.

Knockdown of OVTA1 for the Treatment of Ovarian Cancer

After further knockdown studies, the invention found that the knockdownof OVTA1 could significantly reduce cell growth and cell migration aswell as tumor growth rate. The knockdown of the antigen polypeptide ofthe invention or OVTA1 can be used in the prevention and/or treatment ofovarian cancer and inhibition of metastasis of ovarian cancer.Accordingly, the invention provides a method for the prevention and/ortreatment of ovarian cancer, comprising the step of suppressing theexpression of the antigen polypeptide of the invention or OVTA 1.

EXAMPLE Example 1 Production of the Antigen Polypeptide of the Inventionand Confirmation of the Antigen as an Immunogenic Target

Cell culture. OVCAR-3, SKOV-3, and TOV-112D ovarian cancer cell linesand HeLa were obtained from American Type Culture Collection (Rockville,Md.). OVCAR-3 and SKOV-3 cells were cultured in DMEM and Ham's F-12medium (1:1) (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetalbovine serum (FBS), penicillin (100 units/ml)/streptomycin (100 μg/ml),0.1 mM nonessential amino acids and 1 mM sodium pyruvate (Invitrogen).TOV-112D cells were cultured in MCDB105 and Media 199 (1:1) (Sigma, St.Louis, Mo.) plus 15% FBS, penicillin, and streptomycin. HeLa cells weremaintained in DMEM containing 10% FBS.

Ascetic fluids, serum and tissue samples. 12 ascetic fluid and 36 tissuesection samples were collected under permission of the InstitutionReview Board, National Health Research Institute. Histological subtypesof ascetic samples were 10 serous, 1 clear-cell, and 1 mucinoussubtypes, while the tissue samples included 16 serous-, 9 endometrioid-,6 clear-cell, 4 mucious-, and 1 endometrioid/clear cell mix-subtypes.The control sera were obtained from 10 age-matched healthy women. Theirimmunoglobulin G (IgG) fraction of the ascetic fluid and serum sampleswas purified with immobilized Protein A/G (Pierce, Rockford, Ill.)according to the manufacturer's instructions.

Screening of phage library. Serological selection of phage clonesdisplaying peptides recognized by malignant ascetic antibodies ofpatients with ovarian cancer was performed using a Ph.D.-7 phage displaypeptide system (New England BioLabs, Beverly, Mass.). A bio-panningprocedure was carried out according to the manufacturer's instructionswith certain modifications. Briefly, after removal of non-specificbinders with healthy control IgG mixture, phages (10¹¹ pfu) wereincubated with a pool of IgG antibodies (100 μg/ml) purified from 32patients with ovarian cancer which was pre-coated in a 96-well plate for2 hrs at room temperature. The unbound phages were removed by washing 10times with TBST solution [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1%Tween-20]. The bound phages were eluted in an elution buffer [0.2 MGlycine-HCl (pH 2.2), 1 mg/ml BSA] and propagated by infecting E. colihost cell (strain ER 2738) at 37° C. with vigorous agitation for 4-5hrs. After centrifugation, the cells were discarded. The amplifiedphages were collected by addition of ⅙ volume of PEG/NaCl solution [20%(w/v) polyethylene glycol-8000, 2.5 M NaCl] to the supernatant followedby centrifugation at 4° C., 16,000×g for 15 min. The resultant phageswere re-suspended in TBS [50 mM Tris-HCl (pH 7.5), 150 mM NaCl] and thetiters were determined on LB/IPTG/Xgal plates as described in theprovided manual. The above bio-panning procedure was repeated 3 moretimes to enrich the antibody-bound phage clones.

The peptide sequences presented by bound phages were resolved by directDNA sequencing. Phages from fifteen blue plaques were randomly selected,amplified in E. coli host cells, and collected by precipitation andcentrifugation as described above. The resultant pellets werere-suspended in 100 μl Iodide buffer [10 mM Tris-HCl (pH 8.0), 1 mMEDTA, 4 M NaI] containing 250 μl ethanol. After incubation for 10 min,the single-stranded phage DNA was preferentially precipitated anddissolved in 30 μl TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. TheDNA inserts coding for displayed peptide was determined by directsequencing in an automated DNA sequencer ABI 8700.

Enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed thatone consensus motif, X₁-P-H-X₂-Y-X₃-X₄, was displayed by 87% (13/15) ofthe selected phage clones. To determine the binding of phage containingPeptide #10 to individual antibodies purified from 12 patients withovarian cancer, ELISA assay was performed by coating the purifiedantibodies in each well. After washing, the bound phages were detectedby anti-M13 antibody conjugated with horse-radish peroxidase (HRP)(1:2000) and visualized by incubation with ABTS peroxidase substrate(KPL, Caithersburg Md.). The titers of individual samples were measuredat OD₄₅₀. The control wells were coated with 10 μg/ml of BSA or 100μg/ml purified IgG mixture or sera from healthy subjects. M13 phage notdisplaying any peptide was also included as a negative control.

Gene construction and antiserum generation. The FJ10213 plasmid wasserved as a gene template for coding for KIAA0999 protein in this study.The gene insert was cut with Sal I and Not I enzymes, blunted, andsubcloned into pcDNA3.1 vector at EcoR V, named as pcDNA-FJ10213. On thebasis of the gene sequence of FJ10213 available on NCBI GenBank(accession number: AB023216), an myc-tagged plasmid, named myc-CT-OV1,was constructed by cloning the nucleotides 2278 to 3336 of FJ10213 cloneinto pBlueScript vector with EcoRI sites. This DNA insert encoded a 353amino-acid C-terminal partial protein of OVTA1, including the consensusmotif, TPHGYAH, recognized by the phage clones. The plasmid myc-CT-OV1del-7mer lacking the consensus sequence was generated by site-directmutagenesis using a pair of primers listed below.

Forward primer: (SEQ ID NO: 6) 5′-GCT TCC TCA CCC ACC CCG CAG CCG GCACTG ATG CAT-3′ Backward primer: (SEQ ID NO: 7) 5′-ATG CAT CAG TGC CGGCTG CGG GGT GGG TGA GGA AGC-3′

For generation of an antiserum specific to OVTA1, the nucleotides 2863to 3336 (SEQ ID NO:4) were cloned into a pGEX-KG vector at 5′-Xba I and3′-Xho I sites and expressed in E. coli (JM109 strain). The GST-taggedrecombinant protein was purified as described in a previous study(Clinical Cancer Research, Oct. 1, 2006; 12(19): 5746-5754) and servedas an immunization antigen for rabbits or mice. The resultant antiserawere purified with Melon gel IgG purification system (PierceBiotechnology, Inc., IL, U.S.A.) according to the manufacturer'sinstructions. For functional study in a cell, the full-length OVTA1 genewith nucleotides 438 to 4229 (SEQ ID NO:1) was constructed into avector, pcDNA3-myc, and expressed as an Myc-OVTA1 recombinant protein.Additionally, a plasmid, named pshOVTA1 (clone number: RHS3979-9604860),expressing small interfering RNA to knockdown OVTA1 level and itsscramble control pLKO.1 were obtained from Open Biosystems (Huntsville,Ala., U.S.A.). Stable elevation of OVTA1 gene expression was establishedin OVCAR-3 cells; meanwhile, attenuation of its expression bytransfection of pshOVTA1 was manipulated in SKOV-3 cells.

An attempt was made to select more immunogenic tumor-associated antigensthat could be recognized by autoantibodies purified from ascetic fluidsof patients with ovarian cancer. A 7-mer random peptide phage displaylibrary was used for this screening. The bio-panning experiment for thisscreening is schematically illustrated in FIG. 1. The library waspre-absorbed with total immunoglobin G (IgG) purified from 30 healthydonors to mask peptide epitopes that were recognized by the IgG.Subsequently, the individual purified ascetic IgG obtained from 32patients with ovarian cancer was used to select phages displayingpotential tumor-associated peptides. After 4 bio-panning enrichmentcycles, 13 out of 15 phage clones displayed consensus xPHxYxx sequencesas shown in Table 1 below.

TABLE 1

Consensus: X₁ P H X₂ Y X₃ X₄To reveal displayed peptides of antibody-bound phages, the insertednucleotide sequences of coding for the displayed peptides were directlysequenced. As a result, the displayed peptide sequences of 15 randomlychosen antibody-bound phages after 4^(th) panning are deduced and listedin Table 1. Intriguingly, one consensus motif, X₁PHX₂YX₃X₄, was obtainedfrom those displayed peptide sequences. Putative proteins, found in asearch of NCBI protein database, containing those displayed peptidesequences are indicated in the right column of Table 1. Among them, onehuman novel gene containing Peptide #10 is temporarily named OVTA1 gene,which was previously identified as KIAA0999 gene. The putativepolypeptide encoded by KIAA0999 gene contains Pro-His-Gly-Tyr-Ala-Hissequence. Thus, it suggests that displayed peptide sequences by thosephages may be associated with tumor antigens. In addition, the datashown in FIG. 2 indicates that the phage clone displaying peptideTPHGYAH can strongly bind to ascetic antibodies purified from ovariancancer patient 11 (CA502).

Serological confirmation of the antigen polypeptide of the invention asan immunogenic target. To verify that the antigen polypeptide of theinvention was the immunogenic target of Patient 11's (CA502) asceticantibody, a putative 3′-coding region of OVTA1 gene coding for anMyc-tagged 40 KDa polypeptide containing the Pro-His-Gly-Tyr-Ala-Hissequence was cloned out from SK-OV3 cells. Western blotting analyses(see Example 2 below) of HeLa cells overexpressing the putativeC-terminal OVTA1 (CT-OVTA1) gene tagged with myc sequence and probedwith either purified Patient 11 or anti-Myc antibodies indicated thatthe Peptide #10 containing C-terminus of the invention was indeed animmunogenic target of Patient 11's ascetic autoantibody (FIG. 3A). Tofurther confirm whether the Pro-His-Gly-Tyr-Ala-His sequence was themajor immunogenic epitope for the antibody, a deletion mutant lackingthis particular sequence displayed by the cloned phages was generated.Intriguingly, removal of the sequence totally abolished recognition ofthe Patient autoantibody to the antigen polypeptide of the invention(FIG. 3B), suggesting that this particular sequence in the antigenpolypeptide of the invention is one of the highly immunogenic epitopesfor tumor immunosurveillance.

Example 2 Expression of the Antigen Polypeptide of the Invention

Western blot analysis. The cell lysates were prepared as described in aprevious study (BMC Molecular Biology 2007, 8:72 pp. 1-15). The cellswere transfected with a plasmid coding for wild-type, mutant KIAA0999gene, or control vector using Lipofectamine (Invitrogen Co., CA, U.S.A.)for HeLa cells or Lipofectamine 2000 transfect reagent for OVCAR-3 andSKOV-3 cells according to the manufacturer's instructions. After 24 hrstransfection, cells were lysed in a lysis buffer [20 mM Tris-HCl, 150 mMNaCl, 0.1% SDS, 5 mM MgCl₂, 10% glycerol, 0.5% NP-40, 100 mM NaF, 1 MNa₃VO₄, and protease inhibitor cocktail (Roche Diagnostics,Indianapolis, Ind., U.S.A.)]. The protein concentration was quantifiedusing BCA protein assay (Pierce). Approximately 15 or 150 μg of eachsample was resolved in 7.5-10% SDS-PAGE, transferred to nitrocellulosemembrane, and blotted with an antibody specific to myc-tag (1:10,000dilution), GST-tag (1:1,000 dilution) (Pierce), OVTA1 (1:1,000dilution), or with CA502 purified ascetic antibody (32 μg/ml). Theimmuno-complexes were detected by probing with anti-mouse, -rabbit, or-human IgG conjugated with HRP (Jackson ImmunoResearch, Cambridgeshire,UK) as visualized using the SuperSignal chemiluminescence detectionsystem (Pierce, Rockford, Ill.).

Characterization of OVTA1 transcript and protein in ovarian cancercells. To determine the actual sizes of naturally occurring transcriptand its encoded polypeptide of OVTA1 in ovarian cells, Northern andWestern blotting analyses were carried out. Using an antisense probeembracing the sequence coding for the 7-mer peptide, Northern blottinganalysis clearly indicated a ˜4.5 kb band as the major correspondingtranscript for OVTA1 gene in SK-OV3 and TOV-112D cells while showing asignificantly low level of the transcript in OVCAR3 cells (FIG. 4).Simultaneously, a recombinant CT-OVTA1 gene was constructed that wasfused with GST tag sequence, and the recombinant antigen was expressedand purified for generation of OVTA1-specific rabbit antiserum. Thespecificity of the antiserum has been confirmed by Western blottinganalysis of HeLa cells expressing either myc-tagged CT-OVTA1 gene orvector control (data not shown). Consistently, the molecular weight of amajor immuno-reactive protein is around 150 KDa in SK-OV3 and TOV-112Dcells, but rarely detected in OVCAR3 cells (FIG. 5). The experimentusing an in vitro transcript and translation couple system demonstratedthat the putative KIAA0999 gene (i.e., OVTA1 gene in this invention)published in NCBI database (Access number: AB023216) could be translatedinto a 150 KDa-polypeptide chain (FIG. 5) and be immunoreactive to OVTA1antiserum (data not shown). Thus, taken together, these data clearlyindicate that OVTA1 gene is a functional allele and encode a 150 KDaprotein in ovarian cancer cells.

Tissue distribution of OVTA1 expression. To determine OVTA1 geneexpression in a variety of tissues, we performed polymerase chainreaction (PCR) of cDNA libraries from 12 normal human tissues using twopairs of gene-specific primer located in the 3′-coding and -untranslatedregions of OVTA1 gene. Nested PCR analyses shown in FIG. 6 indicatedthat OVTA1 universally existed in multiple tissues with a distinctdifference in its expression level. Relatively higher levels of itsexpression are observed in the lungs, heart, and testes, whereassignificantly low levels are observed in the brain and ovaries.

Example 3 Increase in Cell Proliferation Rate and Cell Migration byOver-Expression of OVTA1

Over-expression of OVTA1 in OVCAR3 cells Establishment of OVCAR3 StableClones: OVTA1 gene was constructed from SK-OV3 ovarian cancer cells byusing RT-PCR (sense, 5′-CGG GAA TTC TCT CAG CAA CAT GCC AGG C-3′ (SEQ IDNO:23); antisense, 5′-AAT GAA TTC TCA GTT GAT TAG GGC AGA-3′ (SEQ IDNO:24)). The pcDNA-OVTA1 plasmid was constructed by using the followingspecific primers: 5′-CGC CAG TGT GCT GGA ATT CTG CTG TCC GGC GCG AGC-3′(SEQ ID NO:25); antisense, 5′-GCA TGC TCG AGC GGC CGC TTA CAC GCC TGCCTG CTC CAT-3′ (SEQ ID NO:26). OVCAR3 cells were transfected with 1 μgof pcDNA-OVTA1 plasmid for the establishment of OVTA1 stable clones orwith pcDNA3.1 for the vector controls. The stable clones were selectedfrom the medium containing 400 μg/ml of G418 after 4 weeks. Theexpression levels were confirmed by RT-PCR and Western blot analysis.

Expression of OVTA1 leads to cell proliferation and migration in ovariancancer cells It is known that SK-OV3 shows a higher cell proliferationrate in vitro compared to OVCAR3 cells. To investigate whether OVTA1antigen was associated with ovarian tumorogenesis, SK-OV3 cells wereeither transfected with pLKO.1-shOVTA1 or with pLKO.1 vector as a mockcontrol. Meanwhile, OVCAR3 cells were transfected with pcDNA orpcDNA-OVTA1 vector. RT-PCR and Western blot were performed to show thatboth OVTA1 mRNA and protein levels increased in pcDNA-OVTA1 transfectedOVCAR3 cells (FIG. 7).

Migration assay. The cell migration experiments were performed using invitro transwell assays. 5×10⁴ cells were resuspended in serum-freemedium and seeded in a culture insert containing membrane with 8 μm poresize (Costar, Cambridge, Mass., USA). After placing the insert onto awell pre-filled with M199 medium plus 10% FBS, the cells were culturedat 37° C. for 6 hours. The number of the cells migrating from the upperchamber into the bottom side of the insert by chemo-attraction wascounted as described by manufacturer's instruction (Costar).

Consequently, the over-expression of OVTA1 in OVCAR3 cells significantlyincreased cell proliferation rate (FIG. 8A) and cell migration(metastasis) (FIG. 8B) by around 2.75 to 3.1 times compared to control.

Example 4 Knockdown of OVTA1

Knockdown of OVTA1 by small interfering RNA in SK-OV3 cells. ThepLKO.1-shOVTA1 vector containing5′-CCGGGCCAGGCTTTATCTTATCAAACTCGAGTTTGATAAGATAAAGCCTGGCTTTTTG (SEQ IDNO:27) and pLKO.1 control vector (Open Biosystems) were prepared. SK-OV3cells were transfected with 1 μg of pLKO.1-shOVTA1 or pLKO.1 as control.The stable clones were selected by 2.5 μg/ml of puromycin for 4 weeksand the efficiency of interference was analyzed using RT-PCR and Westernblotting. The RT-PCR and Western blot analysis showed that OVTA1 mRNAand protein expressions were suppressed in SK-OV3 cells transfected withpLKO.1-shOVTA1 in the knockdown experiments (FIG. 9). As a result, thesuppression of OVTA1 expression in SK-OV3 cells significantly inhibitedcell proliferation rate (FIG. 10A) and cell migration (metastasis) (FIG.10B). Taken together, these data suggest that high levels of OVTA1 maypromote tumor cell proliferation and metastasis in human ovarian cancercells.

Example 5 Correlation Between OVTA1 Expression and Tumor Progression

Xenograft Mouse Models of Ovarian Cancer. Five- to six-week-old SCIDmice were purchased from the Animal Center of Tzu Chi University,Hualien, Taiwan. Animals were maintained under specific pathogen-freeconditions and provided with sterile food and water. The mice wereallowed to acclimatize for at least six days. SK-OV3 and transfectantswith pLKO.1-shOVTA1 or vector control were subcutaneously injected intothe mice (1×10⁶ cells per mouse in 0.1 ml of phosphate buffered salineunder aseptic conditions). Tumor sizes were observed and recorded weeklyby measuring tumor length (L) and width (W) using a caliper. Tumorvolume was calculated from measurements of width and length as: tumorvolume (width/2)2×length×π (reference).

OVTA1 expression is correlated with tumor progression. To furtherinvestigate the role of OVTA1 in vivo, SCID mice were subcutaneouslyinjected with SK-OV3 cells transfected with pLKO.1-shOVTA1 or controlvector. The tumor growth in each mouse was monitored weekly. The tumorsizes were measured for a total of 8 weeks after cell inoculation.Tumors developed in all the mice subcutaneously inoculated with SK-OV3cells but not in the mice inoculated with SK-OV3/shOVTA1 cells (FIG.11A). The knockdown of OVTA1 decreases tumor growth rate in NOD-SCIDmice (FIG. 11B). These observations strongly suggest that the OVTA1plays a critical role in tumor progression.

Example 5 Use of the Antigen Polypeptide of the Invention as Target ofDiagnosis of Ovarian Cancer

Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue sampleswere obtained from cooperating hospitals and stained with anti-GSTcontrol antibody or antibody specific to CA125 (Dako, Carpinteria,Calif.) or OVTA1 using a conventional method. The sections were seriallydewaxed, re-hydrated, and washed with PBS. After 20 min heat-mediatedantigen retrieval process in 10 mM sodium citrate (pH 6.0), the sectionswere rinsed with a wash buffer [10 mM Tris-HCl (pH 7.4) and 150 mMsodium chloride] three times followed by treatment with 3% hydrogenperoxide for 5 min to block endogenous peroxidases. After PBS wash, thesamples were incubated with the diluted primary antibody (1:20 forCA125, 1:4000 for OVTA1 rabbit antiserum, or 1:2000 dilution forhome-made rabbit anti-GST antiserum) for 1 hour at room temperature orovernight at 4° C. The primary antibodies were detected using LSAB kit(Dako) and the slides were counter-stained with hematoxylin. Theexpression statuses of OVTA1 and CA125 were accessed and graded at afinal magnification of 200× independently by two pathologists in ablinded manner. Conflicting scores were resolved with a discussionmicroscope.

OVTA1 over-expression in ovarian cancer tissues. 40 tissue sections fromnon-cancer ovary and 39 from ovarian cancer tissues were prepared andexamined for their OVTA1 and CA125 expression using either anti-OVTA1 orconventional anti-CA125 antibodies. The immunohistochemic resultsclearly show that OVTA1 is over-expressed in ovarian cancer tissuescompared to normal ovarian tissues or tissues from adenomyosis. Incontrast, CA-125 antigen can be detected in both ovarian cancer tissuesand tissues from adenomyosis. A representative result is shown in FIG.12. The statistical results are summarized in Table 2 below, andindicate that OVTA1 antigen was exclusively detected in ovarian cancerbut not in non-cancer tissues. In sum, it is suggested that OVTA1antigen is a better diagnostic target than CA125, which is routinelyused for the detection of ovarian cancer.

TABLE 2 CA-125 OVTA-1 Subtype Total Metastasis Positive Negative TracePositive Negative Trace Non-ovarian cancer Normal ovary 20 2 16 2 1 19 0Adenomyosis 10 7 2 1 0 10 0 Leiomyoma 10 2 8 0 0 10 0 Total 40 11(27.5%) 26 (65%) 3 1 (2.5%) 39 (97.5%) 0 Ovarian cancer Serous 13 Yes 1212 0 0 9 1 2 No 1 0 1 0 0 1 0 Endometriod 8 Yes 5 5 0 0 3 2 0 No 3 3 0 00 3 0 Clear cell 6 Yes 1 1 0 0 1 0 0 No 5 3 2 0 2 3 0 Mucinous 3 Yes 1 10 0 0 1 0 No 2 0 2 0 0 2 0 Yolk sac 1 Yes 1 0 1 0 0 1 0 No 0 0 0 0 0 0 0Mixed 2 Yes 2 2 0 0 0 2 0 No 0 0 0 0 0 0 0 Others 6 Yes 6 4 2 0 3 2 1 No0 0 0 0 0 0 0 Total Yes 28 25 3 0 16 9 3 No 11 6 5 0 2 9 0

1. An isolated antigen polypeptide expressed in a subject with ovariancancer, selected from the group consisting of: (a) a first polypeptideconsisting of a first amino acid sequence consisting of at least 85%sequence identity with SEQ ID NO:2; (b) a second polypeptide encoded bya first polynucleotide consisting of a nucleotide sequence consisting ofat least 85% sequence identity with SEQ ID NO: 1; and (c) a thirdpolypeptide consisting of a first amino acid fragment encoded by asecond polynucleotide as shown in SEQ ID NO: 4 or consisting of a secondamino acid fragment as shown in SEQ ID NO: 3, provided that the sequenceof the third polypeptide is included within (a) or (b).
 2. The isolatedantigen polypeptide of claim 1, wherein the sequence identity in (a) and(b) is at least 90%, 95% or 98%.
 3. The isolated antigen polypeptide ofclaim 1, which is encoded by a polynucleotide consisting a nucleotidesequence as shown in SEQ ID NO: 1 or SEQ ID NO:
 4. 4. The isolatedantigen polypeptide of claim 1, which consists the first amino acidsequence as shown in SEQ ID NO:2 or the second amino acid fragment ofSEQ ID NO:3.
 5. A kit for the detection of ovarian cancer in a subjectcomprising the antigen polypeptide as claimed in claim 1.